Topographic regulation of cytoskeletal protein phosphorylation in normal neurons and during neurodegeneration. The major questions relate to the factors that regulate the compartmentalization of cytoskeletal protein phosphorylation in neurons and the mechanisms responsible for deregulation in neurodegenerative disorders. To address this question we are using two experimental approaches (1) a study of local exogenous signals that may activate kinases within axons and (2) an analysis of the nature of macromolecular phosphorylation complexes extracted from cell bodies and axons in mammalian and squid systems. With respect to the first, we have shown that proline directed kinases (MAP kinase and cdk5) are primarily responsible for the extensive axonal phosphorylation of multiple KSP repeat sites in NF-M and NF-H tail domains. We have also demonstrated that these kinases are activated by signal transduction cascades in cell culture and phosphorylate NF-M and NF-H KSP repeats. In the axon one source of exogenous signals are the glia responsible for myelination. Our studies of myelin associated glycoprotein (MAG)- null mice in which glial cells fail to myelinate axons, have shown that the activities of Erk1/2 and cdk5, as well as cytoskeleton protein phosphorylation were down regulated in DRGs of MAG null mice. To further explore the mechanism of this signaling we have established an in vitro culture system of PC12 or DRG cells co-cultured with MAG-transfected COS cells. Here, too, we observed activation of Erk1/2 and cdk5 kinases coupled to NF phosphorylation in neuronal cells. With respect to the second approach, we are using the squid giant axon system to understand the topographic regulation of cytoskeletal protein phosphorylation. Our initial studies have demonstrated differential patterns of cytoskeletal protein phosphorylation between neuronal cell bodies and axons. In the squid giant fiber system, extracted axonal phosphorylation complexes (multimeric complexes of cytoskeletal proteins, kinases, phosphatases and regulators) exhibit active phosphorylation of endogenous substrates whereas similar complexes derived from cell bodies are relatively inactive. To study the nature of this difference, we are evaluating various kinases/phosphatases to determine whether differences in their activities are responsible for this compartment-specific pattern of phosphorylation. After four summers of work we have confirmed that in the squid giant axon system, tyrosine protein phosphatase activity is significantly elevated in the perikarya (cell body) compared to the axonal compartment. CDK5: Expression, regulation and its role in nerve cell function: While cyclin-dependent kinase5(cdk5) belongs to the family of cell cycle-dependent kinase (CDKs), it is active only in post mitotic cells due to its binding with neuron specific proteins of molecular weight 35 and 39 kDa. In brain, cdk5 has been shown to play an important role including survival and apotosis. Since its initial identification and cloning from the rat brain cDNA library in our laboratory, we have studied its regulation and role in nervous system development both in vivo and in vitro extensively. In cdk5-/- mice, we observed the presence of hyperphosphorylated cytoskeletal proteins in swollen brain stem and spinal cord neuronal perikarya. This led us to look for other kinases affected by the absence of cdk5. Since cdk5 is down regulated in p35-/-mice, we used brain extracts from these mice and found that MAPK (Erk1/2) was hyperactivated. Indeed, we have shown that MEK1 is an in vitro and in vivo target for cdk5/p35 phosphorylation; MEK1 catalytic activity was inhibited by phosphorylation at a specific site, Thr 286. These results suggest that cdk5 is involved in cross talk with other signal transduction pathways. In addition we found elevated apoptosis of neurons derived from cdk5(-/-) compared to wild type. Currently we are studying the factors regulated by cdk5 in this process. Our preliminary studies show that cdk5 prevents neuronal apoptosis by negative regulation of c-jun N-terminal kinase. We plan to study the signaling pathways that regulate the expression of cdk5/p35 in apoptosis. p25, a truncated form of p35, overstimulates cdk5 activity, promotes abnormal hyperphosphorylation of cytoskeletal proteins in neuron, and correlates with apoptosis and neurodegenerative disorders such as Alzheimers disease. Stress related factors promote this deregulation by favoring proteolytic cleavage of p35 to p25. It is important to reconcile, on the one hand, cdk5/p35 inhibition of apoptosis via its down regulation of the JNK pathway and its apparent upregulation of apoptosis when coupled to p25. p35 is the most effective activator of cdk5, both in vitro and in vivo. To better understand the mechanism of activation we have taken a kinetic approach to study the interaction between p35, or its various truncated forms, with cdk5. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar maximum kinase rates, while cdk5 complexed with full length p35 or with a further truncated form spanning amino acid residues from 138 to 291, (p16) produced slightly less activation ( 80 %) compared to p25. A further truncation peptide that lacks both the essential N- and C-terminal domains exhibits markedly higher affinity for cdk5 than peptides retaining either one of these domains and behaves, in vitro, as a most active inhibitor. We will examine its ability to inhibit specifically the cdk5 activity in vitro as well as in vivo with respect to other members of cyclin-dependent kinases.